This invention relates to methods for increasing the number of copies of a specific nucleic acid sequence or xe2x80x9ctarget sequencexe2x80x9d which may be present either alone or as a component, large or small, of a homogeneous or heterogeneous mixture of nucleic acids. The mixture of nucleic acids may be that found in a sample taken for diagnostic testing, environmental testing, for research studies, for the preparation of reagents or materials for other processes such as cloning, or for other purposes.
The selective amplification of specific nucleic acid sequences is of value in increasing the sensitivity of diagnostic and environmental assays while maintaining specificity; increasing the sensitivity, convenience, accuracy and reliability of a variety of research procedures; and providing ample supplies of specific oligonucleotides for various purposes.
The present invention is particularly suitable for use in environmental and diagnostic testing due to the convenience with which it may be practiced.
The detection and/or quantitation of specific nucleic acid sequences is an increasingly important technique for identifying and classifying microorganisms, diagnosing infectious diseases, detecting and characterizing genetic abnormalities, identifying genetic changes associated with cancer, studying genetic susceptibility to disease, and measuring response to various types of treatment. Such procedures have also found expanding uses in detecting and quantitating microorganisms in foodstuffs, environmental samples, seed stocks, and other types of material where the presence of specific microorganisms may need to be monitored. Other applications are found in the forensic sciences, anthropology, archaeology, and biology where measurement of the relatedness of nucleic acid sequences has been used to identify criminal suspects, resolve paternity disputes, construct genealogical and phylogenetic trees, and aid in classifying a variety of life forms.
A common method for detecting and quantitating specific nucleic acid sequences is nucleic acid hybridization. This method is based on the ability of two nucleic acid strands which contain complementary or essentially complementary sequences to specifically associate, under appropriate conditions, to form a double-stranded structure. To detect and/or quantitate a specific nucleic acid sequence (known as the xe2x80x9ctarget sequencexe2x80x9d), a labelled oligonucleotide (known as a xe2x80x9cprobexe2x80x9d) is prepared which contains sequences complementary to those of the target sequence. The probe is mixed with a sample suspected of containing the target sequence, and conditions suitable for hybrid formation are created. The probe hybridizes to the target sequence if it is present in the sample. The probe-target hybrids are then separated from the single-stranded probe in one of a variety of ways. The amount of label associated with the hybrids is measured.
The sensitivity of nucleic acid hybridization assays is limited primarily by the specific activity of the probe, the rate and extent of the hybridization reaction, the performance of the method for separating hybridized and unhybridized probe, and the sensitivity with which the label can be detected. Under the best conditions, direct hybridization methods such as that described above can detect about 1xc3x97105 to 1xc3x97106 target molecules. The most sensitive procedures may lack many of the features required for routine clinical and environmental testing such as speed, convenience, and economy. Furthermore, their sensitivities may not be sufficient for many desired applications. Infectious diseases may be associated with as few as one pathogenic microorganism per 10 ml of blood or other specimen. Forensic investigators may have available only trace amounts of tissue available from a crime scene. Researchers may need to detect and/or quantitate a specific gene sequence that is present as only a tiny fraction of all the sequences present in an organism""s genetic material or in the messenger RNA population of a group of cells.
As a result of the interactions among the various components and component steps of this type of assay, there is almost always an inverse relationship between sensitivity and specificity. Thus, steps taken to increase the sensitivity of the assay (such as increasing the specific activity of the probe) may result in a higher percentage of false positive test results. The linkage between sensitivity and specificity has been a significant barrier to improving the sensitivity of hybridization assays. One solution to this problem would be to specifically increase the amount of target sequence present using an amplification procedure. Amplification of a unique portion of the target sequence without requiring amplification of a significant portion of the information encoded in the remaining sequences of the sample could give an increase in sensitivity while at the same time not compromising specificity. For example, a nucleic acid sequence of 25 bases in length has a probability of occurring by chance of 1 in 425 or 1 in 1015 since each of the 25 positions in the sequence may be occupied by one of four different nucleotides.
A method for specifically amplifying nucleic acid sequences termed the xe2x80x9cpolymerase chain reactionxe2x80x9d or xe2x80x9cPCRxe2x80x9d has been described by Mullis et al. (See U.S. Pat. Nos. 4,683,195, 4,683,202 and 4,800,159 and European patent applications 86302298.4, 86302299.2, and 87300203.4 and Methods in Enzymology, Volume 155, 1987, pp. 335-350). The procedure uses repeated cycles of primer-dependent nucleic acid synthesis occurring simultaneously using each strand of a complementary sequence as a template. The sequence which is amplified is defined by the locations of the primer molecules that initiate synthesis. The primers are complementary to the 3xe2x80x2-terminal portion of the target sequence or its complement and must complex with those sites in order for nucleic acid synthesis to begin. After extension product synthesis, the strands are separated, generally by thermal denaturation, before the next synthesis step. In the PCR procedure, copies of both strands of a complementary sequence are synthesized.
The strand separation step used in PCR to separate the newly synthesized strands at the conclusion of each cycle of the PCR reaction is often thermal denaturation. As a result, either a thermostable enzyme is required or new enzyme must be added between thermal denaturation steps and the initiation of the next cycle of DNA synthesis. The requirement of repeated cycling of reaction temperature between several different and extreme temperatures is a disadvantage of the PCR procedure. In order to make the PCR convenient, expensive programmable thermal cycling instruments are required.
The PCR procedure has been coupled to RNA transcription by incorporating a promoter sequence into one of the primers used in the PCR reaction and then, after amplification by the PCR procedure for several cycles, using the double-stranded DNA as template for the transcription of single-stranded RNA. (See, e.g. Murakawa et al., DNA 7:287-295 (1988).
Other methods for amplification of a specific nucleic acid sequence comprise a series of primer hybridization, extending and denaturing steps to provide an intermediate double stranded DNA molecule containing a promoter sequence through the use of a primer. The double stranded DNA is used to produce multiple RNA copies of the target sequence. The resulting RNA copies can be used as target sequences to produce further copies and multiple cycles can be performed. (See, e.g., Burg, et al., WO 89/1050 and Gingeras, et al., WO 88/10315.)
Methods for chemically synthesizing relatively large amounts of DNA of a specified sequence in vitro are well known to those skilled In the art; production of DNA in this way is now commonplace. However, these procedures are time-consuming and cannot be easily used to synthesize oligonucleotides much greater in length than about 100 bases. Also, the entire base sequence of the DNA to be synthesized must be known. These methods require an expensive instrument capable of synthesizing only a single sequence at one time. Operation of this instrument requires considerable training and expertise. Methods for the chemical synthesis of RNA have been more difficult to develop.
Nucleic acids may be synthesized by techniques which involve cloning or insertion of specific nucleic acid sequences into the genetic material of microorganisms so that the inserted sequences are replicated when the organism replicates. If the sequences are inserted next to and downstream from a suitable promoter sequence, RNA copies of the sequence or protein products encoded by the sequence may be produced. Although cloning allows the production of virtually unlimited amounts of specific nucleic acid sequences, due to the number of manipulations involved it may not be suitable for use in diagnostic, environmental, or forensic testing. Use of cloning techniques requires considerable training and expertise. The cloning of a single sequence may consume several man-months of effort or more.
Relatively large amounts of certain RNAs may be made using a recombinant single-stranded-RNA molecule having a recognition sequence for the binding of an RNA-directed polymerase, preferably Qxcex2 replicase. (See, e.g., U.S. Pat. No. 4,786,600 to Kramer, et al.) A number of steps are required to insert the specific sequence into a DNA copy of the variant molecule, clone it into an expression vector, transcribe it into RNA and then replicate it with Qxcex2 replicase.
The present invention is directed to novel methods of synthesizing multiple copies of a target nucleic acid sequence which are autocatalytic (i e., able to cycle automatically without the need to modify reaction conditions such as temperature, pH, or ionic strength and using the product of one cycle in the next one).
The present method includes (a) treating an RNA target sequence with a first oligonucleotide which comprises a first primer which has a complexing sequence sufficiently complementary to the 3xe2x80x2-terminal portion of the target to complex therewith and which optionally has a sequence 5xe2x80x2 to the priming sequence which includes a promoter for an RNA polymerase under conditions whereby an oligonucleotide/target sequence complex is formed and DNA synthesis may be initiated, (b) extending the first primer in an extension reaction using the target as a template to give a first DNA primer extension product complementary to the RNA target, (c) separating the DNA extension product from the RNA target using an enzyme which selectively degrades the RNA target; (d) treating the DNA primer extension product with a second oligonucleotide which comprises a primer or a splice template and which has a complexing sequence sufficiently complementary to the 3xe2x80x2-terminal portion of the DNA primer extension product to complex therewith under conditions whereby an oligonucleotide/target sequence complex is formed and DNA synthesis may be initiated, provided that if the first oligonucleotide does not have a promoter, then the second oligonucleotide is a splice template which has a sequence 5xe2x80x2 to the complexing sequence which includes a promoter for an RNA polymerase; (e) extending the 3xe2x80x2-terminus of either the second oligonucleotide or the first primer extension product, or both, in a DNA extension reaction to produce a template for the RNA polymerase; and (f) using the template to produce multiple RNA copies of the target sequence using an RNA polymerase which recognizes the promoter sequence. The oligonucleotide and RNA copies may be used to autocatalytically synthesize multiple copies of the target sequence.
In one aspect of the present invention, the general method includes (a) treating an RNA target sequence with a first oligonucleotide which comprises a first primer which has a complexing sequence sufficiently complementary to the 3xe2x80x2-terminal portion of the target to complex therewith and which has a sequence 5xe2x80x2 to the complexing sequence which includes a promoter for an RNA polymerase under conditions whereby an oligonucleotide/target complex is formed and DNA synthesis may be initiated, (b) extending the first primer in an extension reaction using the target as a template to give a first DNA primer extension product complementary to the RNA target, (c) separating the first DNA primer extension product from the RNA target using an enzyme which selectively degrades the RNA target; (d) treating the DNA primer extension product with a second oligonucleotide which comprises a second primer which has a complexing sequence sufficiently complementary to the 3xe2x80x2-terminal portion of the DNA primer extension product to complex therewith under conditions whereby an oligonucleotide/target complex is formed and DNA synthesis may be initiated; (e) extending the 3xe2x80x2-terminus of the second primer in a DNA extension reaction to give a second DNA primer extension product, thereby producing a template for the RNA polymerase; and (f) using the template to produce multiple RNA copies of the target sequence using an RNA polymerase which recognizes the promoter sequence. The oligonucleotide and RNA copies may be used to autocatalytically synthesize multiple copies of the target sequence. This aspect further includes: (g) treating an RNA copy from step (f) with the second primer under conditions whereby an oligonucleotide/target sequence complex is formed and DNA synthesis may be initiated; (h) extending the 3xe2x80x2 terminus of the second primer in a DNA extension reaction to give a second DNA primer extension product using the RNA copy as a template; (i) separating the second DNA primer extension product from the RNA copy using an enzyme which selectively degrades the RNA copy; (j) treating the second DNA primer extension product with the first primer under conditions whereby an oligonucleotide/target sequence complex is formed and DNA synthesis may be initiated; (k) extending the 3xe2x80x2 terminus of the second primer extension product in a DNA extension reaction to produce a template for an RNA polymerase; and (1) using the template of step (k) to produce multiple copies of the target sequence using an RNA polymerase which recognizes the promoter. Using the RNA copies of step (1), steps (g) to (k) may be autocatalytically repeated to synthesize multiple copies of the target sequence. The first primer which in step (k) acts as a splice template may also be extended in the DNA extension reaction of step (k).
Another aspect of the general method of the present invention provides a method which comprises (a) treating an RNA target sequence with a first primer which has a complexing sequence sufficiently complementary to the 3xe2x80x2 terminal portion of the target sequence to complex therewith under conditions whereby an oligonucleotide/target sequence complex is formed and DNA synthesis may be initiated; (b) extending the 3xe2x80x2 terminus of the primer in an extension reaction using the target as a template to give a DNA primer extension product complementary to the RNA target; (c) separating the DNA extension product from the RNA target using an enzyme which selectively degrades the RNA target; (d) treating the DNA primer extension product with a second oligonucleotide which comprises a splice template which has a complexing sequence sufficiently complementary to the 3xe2x80x2-terminus of the primer extension product to complex therewith and a sequence 5xe2x80x2 to the complexing sequence which includes a promoter for an RNA polymerase under conditions whereby an oligonucleotide/target sequence complex is formed and DNA synthesis may be initiated; (e) extending the 3xe2x80x2 terminus of the DNA primer extension product to add thereto a sequence complementary to the promoter, thereby producing a template for an RNA polymerase; (f) using the template to produce multiple RNA copies of the target sequence using an RNA polymerase which recognizes the promoter sequence; and (g) using the RNA copies of step (f), autocatalytically repeating steps (a) to (f) to amplify the target sequence. Optionally, the splice template of step (d) may also function as a primer and in step (e) be extended to give a second primer extension product using the first primer extension product as a template.
In addition, in another aspect of the present invention, where the sequence sought to be amplified is present as DNA, use of an appropriate Preliminary Procedure generates RNA copies which may then be amplified according to the General Method of the present invention.
Accordingly, in another aspect, the present invention is directed to Preliminary Procedures for use in conjunction with the amplification method of the present invention which not only can increase the number of copies present to be amplified, but also can provide RNA copies of a DNA sequence for amplification.
The present invention is directed to methods for increasing the number of copies of a specific target nucleic acid sequence in a sample. In one aspect, the present invention involves cooperative action of a DNA polymerase (such as a reverse transcriptase) and a DNA-dependent RNA Polymerase (transcriptase) with an enzymatic hybrid-separation step to produce products that may themselves be used to produce additional product, thus resulting in an autocatalytic reaction without requiring manipulation of reaction conditions such as thermal cycling. In some embodiments of the methods of the present invention which include a Preliminary Procedure, all but the initial step(s) of the preliminary procedure are carried out at one temperature.
The methods of the present invention may be used as a component of assays to detect and/or quantitate specific nucleic acid target sequences in clinical, environmental, forensic, and similar samples or to produce large numbers of copies of DNA and/or RNA of specific target sequence for a variety of uses. These methods may also be used to produce multiple DNA copies of a DNA target sequence for cloning or to generate probes or to produce RNA and DNA copies for sequencing.
In one example of a typical assay, a sample to be amplified is mixed with a buffer concentrate containing the buffer, salts, magnesium, nucleotide triphosphates, primers and/or splice templates, dithiothreitol, and spermidine. The reaction is then optionally incubated near 100xc2x0 C. for two minutes to denature any secondary structure. After cooling to room temperature, if the target is a DNA target without a defined 3xe2x80x2 terminus, reverse transcriptase is added and the reaction mixture is incubated for 12 minutes at 42xc2x0 C. The reaction is again denatured near 100xc2x0 C., this time to separate the primer extension product from the DNA template. After cooling, reverse transcriptase, RNA polymerase, and RNAse H are added and the reaction is incubated for two to four hours at 37xc2x0 C. The reaction can then be assayed by denaturing the product, adding a probe solution, incubating 20 minutes at 60xc2x0 C., adding a solution to selectively hydrolyze the unhybridized probe, incubating the reaction six minutes at 60xc2x0 C., and measuring the remaining chemiluminescence in a luminometer. (See, e.g., Arnold, et al., International Publication No. WO 89/02476, published Mar. 23, 1989, International Application No. PCT/US88/03195, filed Sep. 21, 1988, the disclosure of which is incorporated herein by reference and is referred to as xe2x80x9cHPAxe2x80x9d). The products of the methods of the present invention may be used in many other assay systems known to those skilled in the art.
If the target has a defined 3xe2x80x2 terminus or the target is RNA, a typical assay includes mixing the target with the buffer concentrate mentioned above and denaturing any secondary structure. After cooling, reverse transcriptase, RNA polymerase, and RNAse H are added and the mixture is incubated for two to four hours at 37xc2x0 C. The reaction can then be assayed as described above.
The methods of the present invention and the materials used therein may be incorporated as part of diagnostic kits for use in diagnostic procedures.
Definitions
As used herein, the following terms have the following meanings unless expressly stated to the contrary.
1. Template
A xe2x80x9ctemplatexe2x80x9d is a nucleic acid molecule that is being copied by a nucleic acid polymerase. A template may be either single-stranded, double-stranded or partially double-stranded, depending on the polymerase. The synthesized copy is complementary to the template or to at least one strand of a double-stranded or partially double-stranded template. Both RNA and DNA are always synthesized in the 5xe2x80x2 to 3xe2x80x2 direction and the two strands of a nucleic acid duplex always are aligned so that the 5xe2x80x2 ends of the two strands are at opposite ends of the duplex (and, by necessity, so then are the 3xe2x80x2 ends).
2. Primer, Splice Template
A xe2x80x9cprimerxe2x80x9d is an oligonucleotide that is complementary to a template which complexes (by hydrogen bonding or hybridization) with the template to give a primer/template complex for initiation of synthesis by a DNA polymerase, and which is extended by the addition of covalently bonded bases linked at its 3xe2x80x2 end which are complementary to the template in the process of DNA synthesis. The result is a primer extension product. Virtually all DNA polymerases (including reverse transcriptases) that are known require complexing of an oligonucleotide to a single-stranded template (xe2x80x9cprimingxe2x80x9d) to initiate DNA synthesis, whereas RNA replication and transcription (copying of RNA from DNA) generally do not require a primer. Under appropriate circumstances, a primer may act as a splice template as well (see definition of xe2x80x9csplice templatexe2x80x9d that follows).
A xe2x80x9csplice templatexe2x80x9d is an oligonucleotide that complexes with a single-stranded nucleic acid and is used as a template to extend the 3xe2x80x2 terminus of a target nucleic acid to add a specific sequence. The splice template is sufficiently complementary to the 3xe2x80x2 terminus of the target nucleic acid molecule, which is to be extended, to complex therewith. A DNA- or RNA-dependent DNA polymerase is then used to extend the target nucleic acid molecule using the sequence 5xe2x80x2 to the complementary region of the splice template as a template. The extension product of the extended molecule has the specific sequence at its 3xe2x80x2-terminus which is complementary to the sequence at the 5xe2x80x2-terminus of the splice template.
If the 3xe2x80x2 terminus of the splice template is not blocked and is complementary to the target nucleic acid, it may also act as a primer and be extended by the DNA polymerase using the target nucleic acid molecule as a template. The 3xe2x80x2 terminus of the splice template can be blocked in a variety of ways, including having a 3xe2x80x2-terminal dideoxynucleotide or a 3xe2x80x2-terminal sequence non-complementary to the target, or in other ways well known to those skilled in the art.
Either a primer or a splice template may complex with a single-stranded nucleic acid and serve a priming function for a DNA polymerase.
3. Target Nucleic Acid, Target Sequence
A xe2x80x9ctarget nucleic acidxe2x80x9d has a xe2x80x9ctarget sequencexe2x80x9d to be amplified, and may be either single-stranded or double-stranded and may include other sequences besides the target sequence which may not be amplified.
The term xe2x80x9ctarget sequencexe2x80x9d refers to the particular nucleotide sequence of the target nucleic acid which is to be amplified. The xe2x80x9ctarget sequencexe2x80x9d includes the complexing sequences to which the oligonucleotides (primers and/or splice template) complex during the processes of the present invention. Where the target nucleic acid is originally single-stranded, the term xe2x80x9ctarget sequencexe2x80x9d will also refer to the sequence complementary to the xe2x80x9ctarget sequencexe2x80x9d as present in the target nucleic acid. Where the xe2x80x9ctarget nucleic acidxe2x80x9d is originally double-stranded, the term xe2x80x9ctarget sequencexe2x80x9d refers to both the (+) and (xe2x88x92) strands.
4. Promoter/Promoter Sequence
A xe2x80x9cpromoter sequencexe2x80x9d is a specific nucleic acid sequence that is recognized by a DNA-dependent RNA polymerase (xe2x80x9ctranscriptasexe2x80x9d) as a signal to bind to the nucleic acid and begin the transcription of RNA at a specific site. For binding, such transcriptases generally require DNA which is double-stranded in the portion comprising the promoter sequence and its complement; the template portion (sequence to be transcribed) need not be double-stranded. Individual DNA-dependent RNA polymerases recognize a variety of different promoter sequences which can vary markedly in their efficiency in promoting transcription. When an RNA polymerase binds to a promoter sequence to initiate transcription, that promoter sequence is not part of the sequence transcribed. Thus, the RNA transcripts produced thereby will not include that sequence.
5. DNA-dependent DNA Polymerase
A xe2x80x9cDNA-dependent DNA polymerasexe2x80x9d is an enzyme that synthesizes a complementary DNA copy from a DNA template. Examples are DNA polymerase I from E. coli and bacteriophage T7 DNA polymerase. All known DNA-dependent DNA polymerases require a complementary primer to initiate synthesis. It is known that under suitable conditions a DNA-dependent DNA polymerase may synthesize a complementary DNA copy from an RNA template.
6. DNA-dependent RNA Polymerase (Transcriptase)
A xe2x80x9cDNA-dependent RNA polymerasexe2x80x9d or xe2x80x9ctranscriptasexe2x80x9d is an enzyme that synthesizes multiple RNA copies from a double-stranded or partially-double stranded DNA molecule having a (usually double-stranded) promoter sequence. The RNA molecules (xe2x80x9ctranscriptsxe2x80x9d) are synthesized in the 5xe2x80x2xe2x86x923xe2x80x2 direction beginning at a specific position just downstream of the promoter. Examples of transcriptases are the DNA-dependent RNA polymerase from E. coli and bacteriophages T7, T3, and SP6.
7. RNA-dependent DNA Polymerase (Reverse Transcriptase)
An xe2x80x9cRNA-dependent DNA polymerasexe2x80x9d or xe2x80x9creverse transcriptasexe2x80x9d is an enzyme that synthesizes a complementary DNA copy from an RNA template. All known reverse transcriptases also have the ability to make a complementary DNA copy from a DNA template; thus, they are both RNA- and DNA-dependent DNA polymerases. A primer is required to initiate synthesis with both RNA and DNA templates.
8. RNAse H
An xe2x80x9cRNAse Hxe2x80x9d is an enzyme that degrades the RNA portion of an RNA:DNA duplex. RNAse H""s may be endonucleases or exonucleases. Most reverse transcriptase enzymes normally contain an RNAse H activity in addition to their polymerase activity. However, other sources of the RNAse H are available without an associated polymerase activity. The degradation may result in separation of RNA from a RNA:DNA complex. Alternatively, the RNAse H may simply cut the RNA at various locations such that portions of the RNA melt off or permit enzymes to unwind portions of the RNA.
9. Plus/Minus Strand(s)
Discussions of nucleic acid synthesis are greatly simplified and clarified by adopting terms to name the two complementary strands of a nucleic acid duplex. Traditionally, the strand encoding the sequences used to produce proteins or structural RNAs was designated as the xe2x80x9cplusxe2x80x9d strand and its complement the xe2x80x9cminusxe2x80x9d strand. It is now known that in many cases, both strands are functional, and the assignment of the designation xe2x80x9cplusxe2x80x9d to one and xe2x80x9cminusxe2x80x9d to the other must then be arbitrary. Nevertheless, the terms are very useful for designating the sequence orientation of nucleic acids and will be employed herein for that purpose.
10. Hybridize, Hybridization
The terms xe2x80x9chybridizexe2x80x9d and xe2x80x9chybridizationxe2x80x9d refer to the formation of complexes between nucleotide sequences which are sufficiently complementary to form complexes via Watson-Crick base pairing. Where a primer (or splice template) xe2x80x9chybridizesxe2x80x9d with target (template), such complexes (or hybrids) are sufficiently stable to serve the priming function required by the DNA polymerase to initiate DNA synthesis.
11. Primer Sequences
The sequences of the primers referred to herein are set forth below.
HBV region 2 primers
(+): 5xe2x80x2CACCAAATGCCCCTATCTTATCAACACTTCCGG3xe2x80x2 (SEQ ID NO:24)
(xe2x88x92): 5xe2x80x2AATTTAATACGACTCACTATAGGGAGACCCGAGATTGAGATCTTCTGCGAC3xe2x80x2 (SEQ ID NO:25)
Probe:
(+): 5xe2x80x2GGTCCCCTAGAAGAAGAACTCCCTCG3xe2x80x2 (SEQ ID NO:23)
HIV region 1 primers
(+): 5xe2x80x2AATTTAATACGACTCACTATAGGGAGACAAGGGACTTTCCGCTGGGGACTTTCC3xe2x80x2 (SEQ ID NO:2)
(xe2x88x92): 5xe2x80x2GTCTAACCAGAGAGACCCAGTACAGGC3xe2x80x2 (SEQ ID NO:3)
Probe sequence:
5xe2x80x2GCAGCTGCTTATATGCAGGATCTGAGGG3xe2x80x2 (SEQ ID NO:1)
HIV region 2 primers
(+): 5xe2x80x2AATTTAATACGACTCACTATAGGGAGACAAATGGCAGTATTCATCCACA3xe2x80x2 (SEQ ID NO:5)
(xe2x88x92): 5xe2x80x2CCCTTCACCTTTCCAGAG3xe2x80x2 (SEQ ID NO:6)
Probe sequence:
(xe2x88x92): 5xe2x80x2CTACTATTCTTTCCCCTGCACTGTACCCC3xe2x80x2 (SEQ ID NO:4)
HIV region 3 primers
(+): 5xe2x80x2CTCGACGCAGGACTCGGCTTGCTG3xe2x80x2 (SEQ ID NO:8)
(xe2x88x92): 5xe2x80x2AATTTAATACGACTCACTATAGGGAGACTCCCCCGCTTAATACTGACGCT3xe2x80x2 (SEQ ID NO:9)
Probe:
(+): 5xe2x80x2GACTAGCGGAGGCTAGAAGGAGAGAGATGGG3xe2x80x2 (SEQ ID NO:7)
HIV region 4 primers
(+): 5xe2x80x2AATTTAATACGACTCACTATAGGGAGAGACCATCAATGAGGAAGCTGCAGAATG3xe2x80x2 (SEQ ID NO:11)
(xe2x88x92): 5xe2x80x2CCATCCTATTTGTTCCTGAAGGGTAC3xe2x80x2 (SEQ ID NO:12)
Probe:
(xe2x88x92): 5xe2x80x2CTTCCCCTTGGTTCTCTCATCTGGCC3xe2x80x2 (SEQ ID NO:10)
HIV region 5 primers
(+): 5xe2x80x2GGCAAATGGTACATCAGGCCATATCACCTAG3xe2x80x2 (SEQ ID NO:14)
(xe2x88x92): 5xe2x80x2AATTTAATACGACTCACTATAGGGAGAGGGGTGGCTCCTTCTGATAATGCTG3xe2x80x2 (SEQ ID NO:15)
Probe:
5xe2x80x2GAAGGCTTTCAGCCCAGAAGTAATACCCATG3xe2x80x2 (SEQ ID NO:13)
BCL-2 chromosomal translocation major breakpoint t(14;18) primers
(xe2x88x92): 5xe2x80x2GAATTAATACGACTCACTATAGGGAGACCTGAGGAGACGGTGACC3xe2x80x2 (SEQ ID NO:41)
(+): 5xe2x80x2TATGGTGGTTTGACCTTTAG3xe2x80x2 (SEQ ID NO:42)
Probes:
5xe2x80x2GGCTTTCTCATGGCTGTCCTTCAG3xe2x80x2 (SEQ ID NO:43)
5xe2x80x2GGTCTTCCTGAAATGCAGTGGTCG3xe2x80x2 (SEQ ID NO:44)
CML chromosomal translocation t(9;22) primers
(xe2x88x92): 5xe2x80x2GAATTAATACGACTCACTATAGGGAGACTCAGACCCTGAGGCTCAAAGTC3xe2x80x2 (SEQ ID NO:45)
(+): 5xe2x80x2GGAGCTGCAGATGCTGACCAAC3xe2x80x2 (SEQ ID NO:46)
Probe:
5xe2x80x2GCAGAGTTCAAAAGCCCTTCAGCGG3xe2x80x2 (SEQ ID NO:31)
12. Specificity
Characteristic of a nucleic acid sequence which describes its ability to distinguish between target and non-target sequences dependent on sequence and assay conditions.